The Alphavirus genus in the Togaviridae family contains a variety of widely distributed important human and animal pathogens. Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses represent a serious public health threat in the US and can be applied by bioterrorists. However, to date, neither effective antiviral nor safe and efficient vaccines have been developed for preventing any of these infections. The existing experimental vaccines are either of poor efficacies or demonstrate very strong adverse reactions in humans. Our recent studies of the molecular mechanism of alphavirus RNA packaging led to understanding of structure and function of the alphavirus RNA-specific packaging signal, which is present in the genomes of other encephalitogenic alphaviruses and recognized by both homologous and capsid proteins. These data will be applied in the proposed research to develop fundamentally new strategies of designing live alphavirus variants possessing irreversible, highly attenuated phenotypes, but producing virus-specific antigens, required for induction of sterilizing immunity as efficiently as a wt virus. By introducing multiple, rationally designed mutations into capsid protein or newly identified universal RNA packaging signal, we will develop alphaviruses that retain high levels of RNA replication, and production of structural proteins which are released either mostly (strategy 1) or exclusively (strategy 2) in the form of non-infectious, genome-free virus-like particles and, thus, develop no viremia in vivo. These viruses will be capable of inducing a balanced combination of cellular immune response and high levels of neutralizing antibodies. PUBLIC HEALTH RELEVANCE: The research proposal is aimed to develop fundamentally new strategies of designing live alphavirus vaccines demonstrating irreversible, highly attenuated phenotype, but producing virus-specific antigens, required for induction of sterilizing immunity as efficiently as wt viruses.